Methods and systems for assessing biological material using optical detection techniques

ABSTRACT

Optical detection techniques for the assessment of the physiological state, health and/or viability of biological materials are provided. Biological materials which may be examined using such techniques include cells, tissues, organs and subcellular components. The inventive techniques may be employed in high throughput screening of potential diagnostic and/or therapeutic agents.

REFERENCE TO PRIORITY APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/326,244, filed Jun. 4, 1999, issuing as U.S. Pat. No.6,834,238 on Dec. 21, 2004, which claims priority from U.S. ProvisionalPatent Application No. 60/088,494, filed Jun. 8, 1998, entitled METHODSAND APPARATUS FOR ASSESSING BIOLOGICAL MATERIALS USING OPTICAL DETECTIONTECHNIQUES, which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The methods and systems of the present invention employ optical, orspectroscopic, detection techniques for assessing the health,physiological condition, and viability of biological materials such astissues, cells, and subcellular components, and may be used in both invitro and in vivo systems. One important application of the methods andapparatus of the present invention is high throughput screening ofcandidate agents and conditions to evaluate their suitability asdiagnostic or therapeutic agents.

BACKGROUND OF THE INVENTION

Drug development programs rely on in vitro screening assays andsubsequent testing in appropriate animal models to evaluate drugcandidates prior to conducting clinical trials using human subjects.Screening methods currently used are generally difficult to scale up toprovide the high throughput screening necessary to test the numerouscandidate compounds generated by traditional and computational means.Moreover, studies involving cell culture systems and animal modelresponses frequently don't accurately predict the responses and sideeffects observed during human clinical trials.

Conventional methods for assessing the effects of various agents orphysiological activities on biological materials, in both in vitro andin vivo systems, generally are not highly sensitive or informative. Forexample, assessment of the effect of a physiological agent, such as adrug, on a population of cells or tissue grown in culture,conventionally provides information relating to the effect of the agenton the cell or tissue population only at specified points in time.Additionally, current assessment techniques generally provideinformation relating to a single or a small number of parameters.Candidate agents are systematically tested for cytotoxicity, which maybe determined as a function of concentration. A population of cells istreated and, at one or several time points following treatment, cellsurvival is measured. Cytotoxicity assays generally do not provide anyinformation relating to the cause(s) or time course of cell death.

Similarly, agents are frequently evaluated based on their physiologicaleffects, for example, on a particular metabolic function or metabolite.An agent is administered to a population of cells or a tissue sample,and the metabolic function or metabolite of interest is assayed toassess the effect of the agent. This type of assay provides usefulinformation, but it does not provide information relating to themechanism of action, the effect on other metabolites or metabolicfunctions, the time course of the physiological effect, general cell ortissue health, or the like.

Optical techniques have been developed and used for severalapplications. Light scattering has been used in the past to providemeasurements of osmotic water permeability in suspensions of osmoticallyresponsive vesicles and small cells. A. S. Verkman, “Optical Methods toMeasure Membrane Transport Processes,” J. Membrane Biol. 148:99-110,1995. Another study reported a method for the optical measurement ofosmotic water transport in cultured cells. M. Echevarria, A. S. Verkman,“Optical Measurement of Osmotic Water Transport in Cultured Cells: Roleof Glucose Transporters,” J. Gen. Physiol. 99:573-589, 1992.

Optical techniques for observing nerve activity and neuronal tissue arewell-established. Hill and Keynes observed that the nerve from thewalking leg of the shore crab normally has a whitish opacity caused bylight scattering, and that opacity changes evoked by electricalstimulation of that nerve were measurable. Hill, D. K. and Keynes, R.D., “Opacity Changes in Stimulated Nerve,” J. Physiol. 108:278-281,1949. Since the publication of those results, experiments designed tolearn more about the physiological mechanisms underlying the correlationbetween optical and electrical properties of neuronal tissue and todevelop improved techniques for detecting and recording activity-evokedoptical changes have been ongoing.

Intrinsic changes in optical properties of cortical tissue have beenassessed by reflection measurements of tissue in response to electricalor metabolic activity. Grinvald, A., et al., “Functional Architecture ofCortex Revealed by Optical Imaging of Intrinsic Signals,” Nature324:361-364, 1986; Grinvald, et al., “Optical Imaging of NeuronalActivity, Physiological Reviews, Vol. 68, No. 4, October 1988. Grinvaldand his colleagues reported that some slow signals from hippocampalslices could be imaged using a CCD camera without signal averaging.

A CCD camera was used to detect intrinsic signals in a monkey model.Ts'o, D. Y., et al., “Functional Organization of Primate Visual CortexRevealed by High Resolution Optical Imaging,” Science 249:417-420, 1990.The technique employed by Ts'o et al. would not be practical for humanclinical use, since imaging of intrinsic signals was achieved byimplanting a stainless steel optical chamber in the skull of a monkeyand contacting the cortical tissue with an optical oil. Furthermore, inorder to achieve sufficient signal to noise ratios, Ts'o, et al., had toaverage images over periods of time greater than 30 minutes per image.

The mechanisms responsible for intrinsic signals are not wellunderstood. Possible sources of intrinsic signals include dilation ofsmall blood vessels, neuronal activity-dependent release of potassium,and swelling of neurons and/or glial cells caused, for example, by ionfluxes or osmotic activity. Light having a wavelength in the range of500 to 700 nm may also be reflected differently between active andquiescent tissue due to increased blood flow into regions of higherneuronal activity. Yet another factor which may contribute to intrinsicsignals is a change in the ratio of oxyhemoglobin and deoxyhemoglobin inblood.

U.S. Pat. No. 5,215,095 discloses methods and apparatus for real timeimaging of functional activity in cortical areas of a mammalian brainusing intrinsic signals. A cortical area is illuminated, light reflectedfrom the cortical area is detected, and digitized images of detectedlight are acquired and analyzed by subtractively combining at least twoimage frames to provide a difference image. Allowed U.S. patentapplication Ser. No. 08/474,754 discloses similar optical methods andapparatus for optical detection of neuronal tissue and activity.

U.S. Pat. No. 5,438,989 discloses a method for imaging margins, gradeand dimensions of solid tumor tissue by illuminating the area ofinterest with high intensity electromagnetic radiation containing awavelength absorbed by a contrast agent, obtaining a background videoimage of the area of interest, administering a contrast agent, andobtaining subsequent video images that, when compared with thebackground image, identify the solid tumor tissue as an area of changedabsorption. U.S. Pat. No. 5,699,798 discloses methods and apparatus foroptically distinguishing between tumor and non-tumor tissue, and imagingmargins and dimensions of tumors during surgical or diagnosticprocedures.

U.S. Pat. No. 5,465,718 discloses a method for imaging tumor tissueadjacent to nerve tissue to aid in selective resection of tumor tissueusing stimulation of a nerve with an appropriate paradigm activate thenerve, permitting imaging of the active nerve. The '718 patent alsodiscloses methods for imaging of cortical functional areas anddysfunctional areas, methods for visualizing intrinsic signals, andmethods for enhancing the sensitivity and contrast of images. U.S. Pat.No. 5,845,639 discloses optical imaging methods and apparatus fordetecting differences in blood flow rates and flow changes, as well ascortical areas of neuronal inhibition.

U.S. Pat. No. 5,902,732 discloses methods for screening drug candidatecompounds for anti-epileptic activity using glial cells in culture byosomotically shocking glial cells, introducing a drug candidate, andassessing whether the drug candidate is capable of abating changes inglial cell swelling. This patent also discloses a method for screeningdrug candidate compounds for activity to prevent or treat symptoms ofAlzheimer's disease, or to prevent CNS damage resulting from ischemia,by adding a sensitization agent capable of inducing apoptosis and anosmotic stressing agent to CNS cells, adding the drug candidate, andassessing whether the drug candidate is capable of abating cellswelling. A method for determining the viability and health of livingcells inside polymeric tissue implants is also disclosed, involvingmeasuring dimensions of living cells inside the polymeric matrix,osmotically shocking the cells, and then assessing changes in cellswelling. Assessment of cell swelling activity is achieved by measuringintrinsic optical signals using an optical imaging screening apparatus.

SUMMARY OF THE INVENTION

Cells from nearly every organ and tissue, of both plant and animalorigin, can be dissociated into single cells, grown and propagated usingcell culture techniques. Pathological cells from diseased ordysfunctional tissue can also be isolated and maintained in tissueculture. Slices of tissue or tumors may be maintained under cultureconditions for prolonged periods of time and assessed according tomethods of the present invention. Short-term experiments may also beconducted on living acute tissue slices that are prepared and maintainedunder physiological conditions. Many multicellular systems may also bemaintained as functioning systems in cell culture. Healthy, pathogenicand dysfunctional cells and tissue may also be tested and observed insitu in animal models.

All cells undergo physiological processes that contribute to anddetermine their geometrical structure and optical properties. Thesephysiological processes include metabolic processes, volume-regulatoryresponses, gene expression, endocytosis, pinocytosis, ion homeostasis,immune responses, neurological activity and inhibition, responses tomechanical trauma, chemical insult, and the like. Various events,including disease states, dysfunction, inflammation, exposure topathogens, pollutants, radiation, chemotherapy, infectious or otheragents, aging, apoptosis, necrosis, oncogenesis, and the like, affectone or more of these physiological processes, producing measurable andpredictable changes in the characteristic geometrical structure oroptical properties of individual cells and/or cell populations.

The methods and systems of the present invention employ optical, orspectroscopic, detection techniques to assess the physiological state ofbiological materials including cells, tissues, organs, subcellularcomponents and intact organisms. The biological materials may be ofhuman, animal, or plant origin, or they may be derived from any suchmaterials. Static and dynamic changes in the geometrical structureand/or intrinsic optical properties of the biological materials inresponse to the administration of a physiological challenge or a testagent, are indicative and predictive of changes in the physiologicalstate or health of the biological material.

Two different classes of dynamic phenomena are observed in viablebiological materials using optical detection techniques: (1) geometricalchanges in the diameter, volume, conformation, intracellular space ofindividual cells or extracellular space surrounding individual cells;and (2) changes in one or more intrinsic optical properties ofindividual cells or of cell populations, such as light scattering,reflection, absorption, refraction, diffraction, birefringence,refractive index, Kerr effect, and the like. Both classes of phenomenamay be observed statically or dynamically, with or without the aid of acontrast enhancing agent. Geometrical changes may be assessed directlyby measuring (or approximating) the geometrical properties of individualcells, or indirectly by observing changes in the optical properties ofcells. Changes in optical properties of individual cells or cellpopulations may be assessed directly using systems of the presentinvention.

Observation and interpretation of geometrical and/or intrinsic opticalproperties of individual cells or cell populations is achieved in bothin vitro and in vivo systems without altering characteristics of thesample by applying physiologically invasive materials, such asfixatives. Physiologically non-invasive contrast enhancing agents, suchas vital dyes, may be used in desired applications to enhance thesensitivity of optical detection techniques. In applications employingcontrast enhancing agents, the optical detection techniques are used toassess extrinsic optical properties of the biological materials.

Detection and analysis of the geometrical and/or intrinsic opticalproperties of individual cells or sample cell populations providesinformation permitting the classification of the physiological state ofindividual cells or sample cell populations. Based on analysis of thegeometrical and/or optical properties of a sample cell population, thesample may be classified as viable or non-viable, apoptotic, necrotic,proliferating, in a state of activity, inhibition, synchronization, orthe like, or in any of a variety of physiological states, all of whichproduce distinct geometrical and/or optical profiles. The methods andsystems of the present invention therefore provide for identification ofthe physiological state of a sample population and differentiation amongvarious physiological states.

An important application of the methods and systems of the presentinvention involves screening cell populations to assess the effect(s) ofexposure to various types of test agents or test conditions, includingdrugs, hormones and other biological agents, toxins, infectious agents,physiological stimuli, radiation, chemotherapy, and the like. The effectof various test agents and conditions may be evaluated on both normaland pathological sample populations. Safety and cytotoxicity testing isconducted by exposing a sample population to a test agent or testcondition and assessing the physiological state of the sample populationusing optical techniques at one or more time points followingadministration of the test agent or test condition. Such testing may beconducted on various sample populations to determine how a test agent orcondition affects a desired target sample population, as well as topredict whether a test agent or condition produces physiological sideeffects on sample populations that are not the target of the test agentor condition.

According to a preferred embodiment, a disease state or compromisedcondition is simulated in biological materials prior to administrationof a test agent or test condition to assess the suitability of the testagent or condition for treating the disease state or compromisedcondition. Exposure of sample populations to a physiological challenge,such as a change in extracellular osmolarity or ion concentration,altered oxygen or nutrient or metabolite conditions, drugs or diagnosticor therapeutic agents, a disturbance in ion homeostasis, electricalstimulation, inflammation, infection with various agents, radiation, andthe like, simulates a disease state at a cellular or tissue level.Subsequent exposure of the sample populations a test agent or conditionand detection and analysis of changes in geometrical and/or opticalproperties of the sample populations provides information relating tothe physiological state of the sample populations produced by the testagent or condition. Screening techniques may be adapted for use withvarious types of cell sample populations maintained in vitro underappropriate cell culture conditions to provide a high throughput,automated screening system. Alternatively, screening techniques may beadapted to examine cell and tissue populations using various animalmodels to assess the effect of a physiological challenge and/oradministration of a test agent on various cell populations in animalmodels in situ.

Changes in geometrical and/or optical properties of individual cells orcell populations may be determined by reference to empiricallydetermined standards for specific cell types, cell densities and variousphysiological states, or appropriate controls may be run in tandem withthe test samples to provide direct comparative data. Data is collectedand, preferably, stored at multiple time points to provide data relatingto the time course of the effect of a test agent or condition on samplepopulations. Strategies for designing screening protocols, includingappropriate controls, multiple samples for screening various dosages,activities, and the like, are well known in the art and may be adaptedfor use with the methods and systems of the present invention.

DESCRIPTION OF THE FIGURES

Preferred embodiments of the methods and systems for assessingbiological materials using optical detection techniques of the presentinvention will be described with reference to the figures, in which:

FIGS. 1A-1D show a partially schematic flow diagram illustratingexemplary methods and output of the methods and apparatus of the presentinvention with reference to in vitro cell populations, wherein intrinsicoptical properties of an acute rat hippocampal slice maintained in asubmerged perfusion chamber are monitored at intervals during a controlperiod and an activation period, and data is processed according tomethods of the present invention.

FIGS. 2A-2C show the effect of the agent furosemide onstimulation-evoked afterdischarge activity in a hippocampal slicecomparing the field response measurements at an extracellular electrode,with images highlighting changes in optical properties. Experiments wereconducted as described in Example 1.

FIG. 3A illustrates an enlarged grey-scale image of an acute rathippocampal tissue slice, and FIGS. 3B-3E illustrate enlarged acquiredas described in Example 1.

FIG. 4A illustrates a view of human cortex just anterior to face-motorcortex with one recording (R) and two stimulating (S) electrodes, andfour sites (labeled 1, 2, 3, and 4), where average percent changes incorresponding optical properties were determined as described in Example2. FIGS. 4B-4D illustrate plots of the percent optical changes inabsorption in various spatial regions shown in FIG. 4A during electricalstimulation of the human cortex. Experiments were conducted as describedin Example 2.

FIGS. 5A2-5C4 illustrate spatial images of stimulation-inducedepileptiform activity. The images show comparisons between differentdegrees of activation illustrating both the spatial extent and amplitudeof optical changes indicative of the extent of cortical activity.Experiments were conducted as described in Example 2.

FIGS. 6A-6H illustrate percentage difference images in which themagnitude of optical change indicates the regions of greater corticalactivity. Experiments were conducted as described in Example 2.

FIGS. 7A-7H illustrate percentage difference images representing a realtime sequence of dynamic changes of electrical stimulation-evokedoptical changes in human cortex. Experiments were conducted as describedin Example 2.

FIGS. 8A1-8B3 illustrate functional mapping of human language (Broca'sarea) and tongue and palate sensory area in an awake human patient asdescribed in Example 3. FIGS. 8A1 and 8B2 illustrate control percentagedifference images and FIGS. 8A3 and 8B3 illustrate peak optical changeimages following cortical stimulation.

FIGS. 9A and 9B show time course and magnitude plots of dynamic opticalchanges in human cortex evoked in tongue and palate sensory areas and inBroca's area (language). Experiments were conducted as described inExample 3.

FIGS. 10A-10D illustrate the cranial surface of a rat, imaged throughthe intact cranium, and using a contrast enhancing agent to highlightareas of optical change. Experiments were conducted as described inExample 4.

DESCRIPTION OF PREFERRED EMBODIMENTS

The following description of preferred embodiments includes detaileddescriptions of specific applications, as well as specific methods andapparatus. These specific embodiments are described for purposes ofillustrating the scope of the invention; the invention is not limited tothese applications. Techniques for acquiring data relating to opticalproperties of various types of tissues that would be suitable for usewith the methods and systems of the present invention are described innumerous U.S. patents. U.S. Pat. Nos. 5,215,095, 5,438,989, 5,699,798,5,465,718, 5,845,639 and 5,902,732 are hereby incorporated herein byreference in their entireties.

One important application of methods and systems of the presentinvention is to classify the physiological condition or state ofbiological materials based on their geometrical and/or opticalproperties, and to distinguish among various physiological conditions.Viable cell populations are distinguishable from non-viable cellpopulations in both in vitro cell sample populations, and in situ inanimal models based on a comparison of geometrical and/or opticalproperties. Similarly, sample populations that are proliferating, orthat are responding to various stimuli by mounting certain responses,such as immune responses, inflammatory responses, and the like, aredistinguishable from non-responsive sample populations.

Cells or cell populations undergoing apoptosis, an active programmedcell death phenomenon, are likewise distinguishable from cells or cellpopulations undergoing necrosis. Necrosis may result from mechanicalinjury, exposure to toxins, anoxia due to impairment of the bloodsupply, or the like. The physiological changes observed during necrosisinclude swelling, clumping of chromatin, and deterioration of theorganelles, followed by lysis with release of the cell contents, whichare then phagocytized by macrophages. The cytological changes associatedwith apoptosis are very different and include an early condensation ofchromatin and degradation of DNA, and cell volume decreases, with thecell membrane and the organelles remaining intact. Apoptotic cellsultimately fragment into several membrane-bounded globules that arephagocytized by neighboring cells. Neutrophils and macrophages are notinvolved in the terminal stages of apoptotic processes to the sameextent that they are in necrotic processes.

Identifying cell populations undergoing apoptosis is important fornumerous reasons. Certain genes responsible for regulating apoptosisplay a role in cancer, and cancer therapy by irradiation, chemotherapy,and hormone treatment all induce apoptosis in tumor cells. Some cancersmay, in fact, result from the down-regulation of genes that normallycause apoptosis. Hence, methods for screening cell populations toascertain whether or not they are apoptotic is central to gaining aninsight into various pathological conditions, such as cancer. Sincedifferent agents used in cancer treatment induce apoptosis, it is likelythat apoptotic pathways are indicative of the outcome of chemotherapy.

There are many other instances of apoptosis during both normal andpathological cellular activities. For example, hormones regulateapoptosis in gonadal tissues so that numbers and development of spermand egg cells are regulated. In the immune system, apoptosis plays aclear role in the selection of lymphocytes. Immunodeficiency may becaused by lymphocyte developmental blocks which lead to apoptosis bydefault. Cell death in these cases is the normal, programmed response inthe absence of an essential survival signal. However, active inductionof programmed cell death can also elicit immunodeficiency, as inacquired immunodeficiency syndrome, AIDS. Considerable evidence supportsthe proposition that HIV activates T cell apoptosis. In the nervoussystem, apoptosis plays not only a pivotal role during embryogenesis,but also occurs in the adult, generally under various pathologicalconditions that are accompanied by devastating consequences for thepatients. Examples include Alzheimer's disease, amyotrophoic lateralsclerosis (ALS) and other types of neuronal injury inflicted byischemia, hypoglycemia or excitotoxic agents. Trauma, stroke,excitoxicity and hypoxia are conditions of ischemia that are associatedwith extensive neuronal cell death in sensitive brain regions. Recentstudies demonstrated that, after experimentally induced ischemia,characteristic apoptotic DNA fragmentation occurs in affected brainregions. Methods and systems of the present invention for classifyingcell populations as apoptotic, necrotic, viable, non-viable, and thelike, are useful for identifying the physiological state of cells inboth in vitro and in vivo systems, as well as for screening various testagents to ascertain whether they are useful as diagnostic or therapeuticagents.

Methods and systems of the present invention may also be used toidentify physiological conditions associated with and to evaluate testagents and conditions for diagnosis and treatment of various disorders,and pathological conditions, including migraine headaches, spreadingdepression, epilepsy, Alzheimer's disease, multiple sclerosis,psychiatric disorders such as depression, anxiety, bipolar disorder,schizophrenia, Parkinson's disease and other neurodegenerativedisorders, inflammation, trauma, malignancies such as cancer,angiogenesis, wound healing, immune deficiencies, and the like. Testagents and conditions may also be tested for safety and efficacy forapplications such as toxicology, learning and memory, bone growth andmaintenance, muscle and blood systems, sensory-input systems, and thelike.

Optical contrast enhancing agents useful for enhancing the sensitivityof optical detection for various types of cells, physiological states,and the like, may also be screened and identified using methods andsystems of the present invention. Sample populations comprisingmalignant, pathological, or dysfunctional cells may be exposed to testagents, for example, to identify agents that preferentially identify anddistinguish malignant, pathological, and dysfunctional cells or tissue.

According to one embodiment, methods and systems of the presentinvention acquire and compare data representative of one or moredimensional properties of individual cells or cell samples. Acquisition,processing and analysis of data relating to optical properties isdescribed throughout this description. Acquisition of data relating todimensional properties of individual cells is described below.Acquisition and analysis of data relating to dimensional properties maybe achieved using the same or similar methods and apparatus describedherein with reference to optical properties.

In sparsely populated cell samples, cell areas may be approximated usinga single plane of focus. If it is desired to calculate volume, thez-axis (focus) can be automatically adjusted as well. For example, as anautomated and controlled stage moves, the optically transparentcontainer containing the sample population is positioned so that aseries of data sets for multiple, spatially resolved areas of interestcan be acquired, each image being acquired at a predetermined focalplane. The volume for each z-plane can be approximated (see algorithmbelow) and then the volumes for each z-coordinate added together.

General techniques for approximating cell areas and volumes, based onDoughty, S., “Calculating property for solids of revolution,” MachineDesign, pp 184-186, 10 Dec. 1981, are described below. These techniquesare based on Green's theorem: $\begin{matrix}{\oint( {{P{\mathbb{d}x}} + {Q{\mathbb{d}y}}} )} & {\int{\int{( {\frac{\partial Q}{\partial x} - \frac{\partial P}{\partial y}} ){\mathbb{d}x}{\mathbb{d}y}}}} \\({boundary}) & ({area})\end{matrix}$

Individual cells are examined using an appropriate magnifying device.Edge detection of cell boundaries is achieved using, for example, aSobel operator. The boundary is approximated by fitting it to aplurality of straight line segments of “n” line segments by “n” nodes.The integration of the boundary may be taken as “n” line integrals asfollows:∮_(Γ)(…)𝕕s = ∫_(X₁, Y₁)^(X₂, Y₂)(…)𝕕s + ∫_(X₂, Y₂)^(X₃, Y₃)(…)𝕕s + … + ∫_(X_(n), Y_(n))^(X₁, Y₁)(…)𝕕s

There are three cases:

-   -   Case 1: a vertical line, x=constant;    -   Case 2: a horizontal line, y=constant;    -   Case 3: an inclined line, y=5_(i)(x−x_(i))+y_(i)

The area is thus:A=∫∫ _(A)·1dxdy

To apply Green's theorem, the integral can be considered in the form ofδQ/δx−δP/δy and appropriate functions can be devised, e.g., P(x, y) andQ(x, y). For an area calculation, consider Q(x, y)=0 so that φQdy=0, andlet P=−y so that δP/δy=−1. Then, the area can be calculated as follows:$\begin{matrix}{A = {{\int{\int{1\quad{\mathbb{d}x}{\mathbb{d}y}}}} = {- {\int{\int{\delta\quad{P/\delta}\quad y\quad{\mathbb{d}x}{\mathbb{d}y}}}}}}} \\{= {\int{\int{( {{2{Q/{\delta x}}} - {\delta\quad{P/\delta}\quad y}} ){\mathbb{d}x}{\mathbb{d}y}}}}} \\{= {\oint( {{P{\mathbb{d}x}} + {Q{\mathbb{d}y}}} )}} \\{( {{{Green}'}s\quad{Theorem}} )} \\{= {\oint{P{\mathbb{d}x}}}} \\{= {- {\oint{y{\mathbb{d}x}}}}}\end{matrix}$

-   -   Case 2: ΔA=−y_(i)(x_(i+1)−x_(i)) Case 1=0    -   Case 3: ΔA=−[½5_(i)(x² _(i+1)−x_(i) ²)+(y_(i)−5        _(i)x_(i))(x_(i+1)−x_(i))] A=ΣΔA

For volume calculations, conventional edge detection using, for example,a Sobel operator, can be used to focus through an individual cell, whichcan be divided into a plurality (n) of individual, planar sections. Thevolume for each of the “n” sections can be calculated asΔV_(i);=Δz_(i);·ΔA; and the volume of the entire cell can beapproximated as: V=ΣΔV_(i). The determination and comparison of cellareas and volumes is preferably accomplished using computer hardwareand/or software implementations.

According to another embodiment, methods and systems of the presentinvention acquire and compare data representative of one or more opticalproperties of individual cells or areas of interest in cell samplepopulations. Changes in optical properties that are indicative ofphysiological activity and that may be detected include, for example,reflection, refraction, diffraction, absorption, scattering,birefringence, refractive index, Kerr effect, and the like. Changes inoptical properties are detected directly using photon sensitive elementsand, optionally, optical elements that enhance the detected opticalproperties.

High resolution detection of dynamic geometrical and optical propertiesindicative of physiological activity may be accomplished without usingdyes or other types of contrast enhancing agents according to themethods and apparatus of the present invention, as evidenced by theexamples described herein. Many of the assessment techniques andapparatus of the present invention are physiologically noninvasive, inthat detection and analysis of geometrical and/or intrinsic opticalinformation does not require direct contact of the area of interest withany agents such as dyes, oils, devices, or the like. For particularapplications, it may, however, be useful to administer contrastenhancing agents that amplify differences in an optical property beingdetected as a function of physiological activity prior to acquiringsubsequent data and generating a comparison. The use of contrastenhancing agents is described in detail, with reference to opticalimaging of tumor and non-tumor tissue, in U.S. Pat. No. 5,465,718 andU.S. Pat. No. 5,438,989, which are incorporated by reference herein intheir entireties. Suitable contrast enhancing a gents includefluorescent and phosphorescent materials, dyes that bind to cellmembranes, optical probes that preferentially accumulate in blood or inthe intracellular space, phase resonance dye pairs, and the like.Detectors appropriate for use with such contrast enhancing agents arewell known in the art.

Numerous devices for acquiring, processing and displaying datarepresentative of one or more geometrical and/or optical properties of acell sample population in culture or an area of interest in situ in ananimal model may be employed. One preferred device is a camera thatacquires images of one or more areas of interest at predetermined timeintervals that can be compared to identify areas of changes ingeometrical and/or optical properties that indicate physiologicalactivity or dysfunction. The data acquisition device preferablyincorporates or is used in conjunction with a device that magnifies thearea of interest, such as a microscope.

Magnification sufficient to provide resolution of individual cells ispreferred. An inverted microscope such as a Nikon Diophot 300 issuitable. For high throughput screening techniques using cell samplepopulations maintained under culture conditions, samples in opticallytransparent containers such as flasks, plates and multi-well plates, maybe placed on an automated stage that is controlled and moved in aprogrammed fashion to permit periodic examination of individual cells orcell populations according to a programmed schedule. For example, amulti-well culture plate having a plurality of cell samples may beplaced on an automated and controllable microscope stage. The stage iscontrolled by an automated microcontroller so that it automaticallymoves into position over each culture well. A data set relating togeometrical and/or optical properties of individual cells or a cellpopulation is acquired for each position. In this manner, the system canrapidly and systematically acquire data corresponding to many samples.The physiological environment in selected wells may be altered byexposure to a physiological challenge, test agent or test condition, andthe system may continue to automatically acquire data from the samewells in each culture plate at predetermined time intervals followingtreatment, with data acquired from various treatment wells beingcompared to data acquired from various control wells or empiricallydetermined controls.

Acquisition of data representative of one or more geometrical and/oroptical properties preferably provides high spatial resolution as well,so that geometrical or optical data corresponding to a particularspatial location is acquired at various time intervals for comparison.In this fashion, data acquired from single cells or highly localizedareas of interest in cell sample populations is compared to providereliable and highly-sensitive information concerning the physiologicalstate or condition of the sample population. High spatial resolution isprovided, for example, by implementing high resolution cameras andcharge coupled devices (CCDs). Apparatus suitable for obtaining suchimages have been described in the patents incorporated herein byreference and are more fully described below. The optical detectorpreferably provides images having a high degree of spatial resolution ata magnification sufficient to detect single cells. Several images may beacquired at predetermined time intervals and combined, such as byaveraging, to provide images for comparison.

Various data processing techniques may be advantageously used to assessthe data collected in accordance with the present invention. Comparisondata may be assessed or presented in a variety of formats. Processingmay include averaging or otherwise combining a plurality of data sets toproduce control, subsequent and various comparison data sets. Data maybe converted from an analog to a digital form for processing, and backto an analog form for display as an image. Alternatively, data may beacquired, processed, analyzed, and output in a digital form.

Data processing may also include amplification of certain signals orportions of a data set (e.g., areas of an image) to enhance the contrastseen in data set comparisons, and to thereby identify cells or cellpopulations undergoing changes in geometrical and/or optical propertieswith a high degree of spatial resolution. For example, according to oneembodiment, images are processed using a transformation in which imagepixel brightness values are remapped to cover a broader dynamic range ofvalues. A “low” value may be selected and mapped to zero, with all pixelbrightness values at or below the low value set to zero, and a “high”value may be selected and mapped to a selected value, with all pixelbrightness values at or above the high value mapped to the high value.Pixels having an intermediate brightness value, representing the dynamicchanges in brightness indicative of neuronal activity, may be mapped tolinearly or logarithmically increasing brightness values. This type ofprocessing manipulation is frequently referred to as a “histogramstretch” and can be used according to the present invention to enhancethe contrast of data sets, such as images, representing changes inneuronal activity.

Data processing techniques may also be used to manipulate data sets toprovide more accurate combined and comparison data. For example, for invivo applications, movement, respiration, heartbeat, seizure or reflexactivity may shift an area of interest during data acquisition. It isimportant that corresponding data points in data sets are spatiallyresolved and precisely aligned to provide accurate combined andcomparison data. Optical markers may be fixed at an area of interest anddetected as the data is collected to aid in manual alignment ormathematical manipulation of data sets. Various processing techniquesare described below and in the patents incorporated herein by reference.

Comparison data may be displayed in a variety of ways. Comparison datamay be displayed, for example, in a graphical format that highlightsgeometrical or optical differences indicative of physiological changes.A preferred technique for presenting and displaying comparison data isin the form of visual images or photographic frames corresponding tospatially resolved areas of interest. This format provides avisualizable spatial location (two- or three-dimensional) of a cellpopulation being analyzed. To enhance and provide better visualizationof high contrast areas indicating changes in geometrical and/or opticalproperties indicative of physiological activity or dysfunction,comparison data may be processed to provide an enhanced contrast greyscale or even a color image. A look up table (“LUT”) may be provided,for example, that converts the grey scale values for each pixel to adifferent (higher contrast) grey scale value, or to a color value. Colorvalues may map to a range of grey scale values, or color may be used todistinguish between positive-going and negative-going geometrical oroptical changes. In general, color-converted images provide highercontrast images that highlight changes in optical propertiesrepresenting physiological activity, function or dysfunction.

Systems of the present invention generally comprise an illuminationsource for illuminating the biological material, an optical detector foracquiring data relating to a geometrical or optical property of thebiological material, and data storage and analysis and output device(s)for storing data relating to a geometrical or optical property of thebiological material, comparing various data sets, and/or control dataprofiles, to generate comparison data relating to changes in geometricaland/or optical properties indicative of changes in the physiologicalstate of sample populations and to provide or display the output data ina useful format.

An emr source is used for illuminating an area of interest duringacquisition of data representing one or more dimensional or intrinsicoptical properties of cells or tissue at an area of interest. The emrsource may be utilized to illuminate an area of interest directly, aswhen in vitro cell cultures maintained in optically transparentcontainers are illuminated or when tissue is exposed, such as inconnection with surgery, or it may be utilized to illuminate an area ofinterest indirectly through adjacent or overlying tissue such as bone,dura, skin, muscle and the like. The emr source employed in the presentinvention may be a high or low intensity source, and may providecontinuous or non-continuous illumination. Suitable illumination sourcesinclude high and intensity sources, broad spectrum and non-chromaticsources, tungsten-halogen lamps, lasers, light emitting diodes, and thelike. Cutoff filters for selectively passing all wavelengths above orbelow a selected wavelength may be employed. A preferred cutoff filterexcludes all wavelengths below about 695 nm.

Preferred emr wavelengths for acquiring data relating to intrinsicoptical signals include, for example, wavelengths of from about 450 nmto about 2500 nm, and most preferably, wavelengths of the near infraredspectrum of from about 700 nm to about 2500 nm. Generally, longerwavelengths (e.g., approximately 800 nm) are employed to detect cellularor tissue condition of locations beneath the surface of cells or tissue,or beneath other materials such as skin, bone, dura, and the likecortical activity. Selected wavelengths of emr may also be used, forexample, when various types of contrast enhancing agents areadministered. The emr source may be directed to the area of interest byany appropriate means. For some applications, the use of optical fibersis preferred. One preferred arrangement provides an emr source throughstrands of fiber optic using a beam splitter controlled by a D.C.regulated power supply (Lambda, Inc.).

The optical detection methods of the present invention may also usefullyemploy non-continuous illumination and detection techniques. Forexample, short pulse (time domain), pulsed time, and amplitude modulated(frequency domain) illumination sources may be used in conjunction withsuitable detectors (see, Yodh, A. and Chance, B., Physics Today, March,1995). Frequency domain illumination sources typically comprise an arrayof multiple source elements, such as laser diodes, with each elementmodulated at 180° out of phase with respect to adjacent elements (see,Chance, B. et al., Proc. Natl. Acad. Sci. USA, 90:3423-3427, 1993).Two-dimensional arrays, comprising four or more elements in twoorthogonal planes, can be employed to obtain two-dimensionallocalization information. Such techniques are described in U.S. Pat.Nos. 4,972,331 and 5,187,672 which are incorporated by reference hereinin their entireties.

Time-of-flight and absorbance techniques (Benaron, D. A. and Stevenson,D. K., Science 259:1463-1466, 1993) may also be usefully employed in thepresent invention. In yet another embodiment of the present invention, ascanning laser beam may be used in conjunction with a suitable detector,such as a photomultiplier tube, to obtain high resolution data images,preferably in the form of an area of interest.

Illumination with a part of the infrared spectrum allows for detectionof intrinsic optical signals through tissue overlying or adjacent thearea of interest, such as dura and skull. One exemplary infrared emrsource suitable for detection of intrinsic optical signals throughtissue overlying or adjacent the area of interest is a Tunable IR DiodeLaser from Laser Photonics, Orlando, Fla. When using this range of farinfrared wavelengths, the optical detector is preferably provided as aninfrared (IR) detector. IR detectors may be constructed from materialssuch as indium arsenide, germanium and mercury cadmium telluride, andare generally cryogenically cooled to enhance their sensitivity to smallchanges in infrared radiation. One example of an IR detection systemwhich may be usefully employed in the present invention is an IRC-64infrared camera (Cincinnati Electronics, Mason, Ohio).

The area of interest is preferably evenly illuminated to effectivelyadjust the signal over a full dynamic range, as described below.Nonuniformity of illumination is generally caused by fluctuations of theillumination source and intensity variations resulting from thethree-dimensional nature of the tissue surface. More uniformillumination can be provided over the area of interest, for example, byusing diffuse lighting, mounting a wavelength cutoff filter in front ofthe optimal detector and/or emr source, or combinations thereof.Fluctuation of the illumination source itself is preferably prevented byusing a light feedback mechanism to regulate the power supply of theillumination source. In addition, a sterile, optically transparent platemay contact and cover an area of interest to provide a flatter, moreeven contour surface for detection. Fluctuations in illumination can becompensated for using detection processing algorithms, including placinga constant shade grey image marker point at the area of interest as acontrol point.

The system also comprises an optical detector for acquiring a signalrepresentative of one or more optical properties of the area ofinterest. Any photon detector may be employed as an optical detector.Suitable optical detectors include, for example, photo diodes, photomultiplier tubes, photo sensitive silicon detector chips, such as thoseprovided in CCD devices, and the like. Multiple emr sources and/ormultiple photon detectors may be provided and may be arranged in anysuitable arrangement. Specialized detectors for detecting selectedoptical properties may be employed. One preferred optical detector foracquiring data in the format of an analog video signal is a CCD videocamera which produces an output video signal at 30 Hz having, forexample, 512 horizontal lines per frame using standard RS 170convention. One suitable device is a CCD-72 Solid State Camera (Dage-MTIInc., Michigan City, Ind.). Another suitable device is a COHU 6510 CCDMonochrome Camera with a COHU 6500 electronic control box (COHUElectronics, San Diego, Calif.). In some cameras, the analog signal isdigitized 8-bits deep on an ADI board (analog-to-digital board). The CCDmay be cooled, if necessary, to reduce thermal noise.

Data processing is an important feature of the optical detection andanalysis techniques and systems of the present invention. In use, forexample, a CCD apparatus is preferably adjusted (at the level of theanalog signal and before digitizing) to amplify the signal and spreadthe signal across the full possible dynamic range, thereby maximizingthe sensitivity of the apparatus. Specific methods for detecting opticalsignals with sensitivity across a full dynamic range are described indetail in the patents incorporated herein by reference. Means forperforming a histogram stretch of the difference frames (e.g.,Histogram/Feature Extractor HF 151-1-V module, Imaging Technology,Woburn, Mass.) may be provided, for example, to enhance each differenceimage across its dynamic range. Exemplary linear histogram stretches aredescribed in Green, Digital Image Processing: A Systems Approach, VanNostrand Reinhold: New York, 1983. A histogram stretch takes thebrightest pixel, or one with the highest value in the comparison image,and assigns it the maximum value. The lowest pixel value is assigned theminimum value, and every other value in between is assigned a linearvalue (for a linear histogram stretch) or a logarithmic value (for a loghistogram stretch) between the maximum and minimum values. This allowsthe comparison image to take advantage of the full dynamic range andprovide a high contrast image that clearly identifies areas of neuronalactivity or inactivity.

Noise (such as 60 Hz noise from A.C. power lines) is filtered out in thecontrol box by an analog filter. Additional adjustments may furtherenhance, amplify and condition the analog signal from a CCD detector.One means for adjusting the input analog signal is to digitize thissignal at video speed (30 Hz), and view the area of interest as adigitized image that is subsequently converted back to analog format.

It is important that data, such as consecutive data sets correspondingto a particular area of interest, be aligned so that data correspondingto the same spatially resolved location is compared. If data sets aremisaligned prior to comparison, artifacts are introduced and theresulting comparison data set may amplify noise and edge information.Data misalignment may be caused by sample movement or motion, heartbeat,respiration, and the like. Large movements of cells in an area ofinterest being analyzed may require a new orientation of the detector.It is possible to compensate for small movements of cells in the area ofinterest by either mechanical or computational means, or a combinationof both.

Real-time motion compensation and geometric transformations may also beused to align corresponding data. Simple mechanical translation of dataor more complex (and generally more accurate) geometric transformationtechniques can be implemented, depending upon the input data collectionrate and amount and type of data processing. For many types of imagedata, it is possible to compensate by a geometrical compensation whichtransforms the images by translation in the x-y plane. In order for analgorithm such as this to be feasible, it must be computationallyefficient (preferably implementable in integer arithmetic), memoryefficient, and robust with respect to changes in ambient light.

For example, functional control points or numbers can be located in anarea of interest and triangulation-type algorithms used to compensatefor movements of these control points. Goshtasby (“Piecewise LinearMapping Functions for Image Registration,” Pattern Recognition19:459-66, 1986) describes a method whereby an image is divided intotriangular regions using control points. A separate geometricaltransformation is applied to each triangular region to spatiallyregister each control point to a corresponding triangular region in acontrol image.

“Image warping” techniques may be employed whereby each subsequent imageis registered geometrically to the averaged control image to compensatefor movement. Image warping techniques (described in, for example,Wolberg, Digital Image Warping, IEEE Computer Society Press: LosAlamitos, Calif., 1990), may be used. Image warping techniques canfurther indicate when movement has become too great for effectivecompensation and a new averaged control image must be acquired.

The data storage processing and analysis function is generally performedand controlled by a host computer. The host computer may comprise anygeneral computer (such as an IBM PC type with an Intel 386, 486, Pentiumor similar microprocessor or Sun SPARC) that is interfaced with the emrsource and/or optical detector and controls data acquisition and flow,comparison computations, analysis, output, and the like. The hostcomputer thus controls acquisition and analysis of data and provides auser interface.

The host computer may comprise a single-board embedded computer with aVME64 interface, or a standard (IEEE 1014-1987) VME interface, dependingupon bus band width considerations. Host computer boards which may beemployed in the present invention include, for example, ForceSPARC/CPU-2E and HP9000 Model 7471. The user interface can be, forexample, a Unix/X-Window environment. The image processing board can be,for example, based upon Texas Instruments' MVP and other chips toprovide real-time image averaging, registration and other processingnecessary to produce high quality difference images for intraoperativeviewing. This board will also drive a 120×1024 RGB display to show asequence of difference images over time with pseudo-color mapping tohighlight tumor tissue. Preferably, a second monitor is used for thehost computer to increase the overall screen real estate and smooth theuser interface. The processing board (fully programmable) can support aVME64 master interface to control data transactions with the otherboards. Lastly, a peripheral control board can provide electricalinterfaces to control mechanical interfaces from the host computer. Suchmechanical interfaces can include, for example, the light source andoptical detector control box.

A real-time data acquisition and display system, for example, maycomprise four boards for acquisition, image processing, peripheralcontrol and host computer. A minimal configuration with reducedprocessing capabilities may comprise just the acquisition and hostcomputer boards. The acquisition board comprises circuitry to performreal-time averaging of incoming video frames and allow readout ofaveraged frames at a maximum rate bus. A VME bus is preferred because ofits high peak bandwidth and compatibility with a multitude of existingVME products. The acquisition board should also support many differenttypes of optical detectors via a variable scan interface. A daughterboard may support the interfacing needs of many different types ofoptical detectors and supply variable scan signals to the acquisitionmotherboard. Preferably, the unit comprises a daughter board interfacingto an RS-170A video signal to support a wide base of cameras. Othercamera types, such as slow scan cameras with a higher spatial/contrastresolution and/or better signal to noise ratio, can be developed andincorporated in the inventive device, as well as improved daughterboards to accommodate such improved cameras.

Data relating to dimensional and/or intrinsic optical properties of asample population acquired, for example, as analog video signals, may becontinuously processed using, for example, an image analyzer (e.g.,Series 151 Image Processor, Imaging Technologies, Inc., Woburn, Mass.).An image analyzer receives and digitizes an analog video signal with ananalog to digital interface and performs at a frame speed of about{fraction (1/30)}th of a second (e.g., 30 Hz or “video speed”).Processing the signal involves first digitizing the signal into a seriesof pixels or small squares assigned a value (in a binary system)dependent upon the number of photons (i.e., quantity of emr) beingreflected off tissue from the part of the area of interest assigned tothat pixel. For example, in a standard 512×512 image from a CCD camera,there would be 262,144 pixels per image. In an 8 bit system, each pixelis represented by 8 bits corresponding to one of 256 levels of grey.

The signal processor may include a programmable look-up table (e.g.,CM150-LUT16, Imaging Technology, Woburn, Mass.) initialized with valuesfor converting grey coded pixel values, representative of a black andwhite image, to color coded values based upon the intensity of each greycoded value. Using image stretching techniques, the highest and lowestpixel intensity values representing each of the pixels in a digitalimage frame are determined over a region of the image frame which is tobe stretched. Stretching a selected region over a larger range of valuespermits, for example, easier identification and removal of relativelyhigh, spurious values resulting from noise.

The signal processor means may further include a plurality of framebuffers having frame storage areas for storing frames of digitized imagedata received from the A/D interface. The frame storage area comprisesat least one megabyte of memory space, and preferably at least 8megabytes of storage space. An additional 16-bit frame storage area maybe provided as an accumulator for storing processed image frames havingpixel intensities represented by more than 8 bits. The processor meanspreferably includes at least three frame buffers, one for storing theaveraged control image, another for storing the subsequent image, and athird for storing a comparison image.

The signal processor may further comprise an arithmetic logic unit(e.g., ALU-150 Pipeline Processor) for performing arithmetical andlogical functions on data located in one or more frame buffers. An ALUmay, for example, provide image (data) averaging in real time. A newlyacquired digitized image may be sent directly to the ALU and combinedwith control images stored in a frame buffer. A 16 bit result can beprocessed through an ALU, which will divide this result by a constant(i.e., the total number of images). The output from the ALU may bestored in a frame buffer, further processed, or used as an input andcombined with another image.

Normally, areas of increased physiological activity exhibit an increaseof the emr absorption capacity of the cell sample or tissue (i.e., thecell sample gets darker if visible light is used for emr illumination,or an intrinsic signal increases in a positive direction). Similarly, areduction in physiological activity generally corresponds to a decreaseof emr absorption capacity of the tissue (i.e., the tissue appearsbrighter, or intrinsic signals become negative). For example, data set Ais a subsequent averaged image and data set B is an averaged controlimage. Normally, when a pixel in data set A is subtracted from a pixelin data set B and a negative value results, this value is treated aszero. Hence, difference images cannot account for areas of inhibition.The present invention provides a method for identifying both negativeand positive intrinsic signals, by: (a) subtracting data set A (asubsequent averaged image) from data set B (an averaged control image)to create a first difference data set, whereby all negative pixel valuesare zero; and (b) subtracting data set B from data set A to create asecond difference data set whereby all negative pixel values are zero;and adding the first and second difference data sets to create a “sumdifference data set.” The sum difference data set shows areas ofincreased activity (i.e., color coded with warmer colors such as yellow,orange, red) and may be visualized as image areas of less activity orinhibition (i.e., color coded with colder colors such as green, blue,purple). Alternatively, one can overlay the first difference data set onthe second difference data set. The difference output may be visualizedas an image and may be superimposed on the real time analog image toprovide an image of the area of interest (e.g., cortical surface)superimposed with a color-coded difference frame to indicate where thereare intrinsic signals in response to a challenge, stimulus, paradigm, orthe like.

The comparison (e.g., difference) data may be further processed tosmooth out the data and remove high frequency noise. For example, alowpass spatial filter can block high spatial frequencies and/or lowspatial frequencies to remove high frequency noises at either end of thedynamic range. This provides a smoothed-out processed difference dataset (in digital format). The digitally processed difference data set canbe provided as an image and color-coded by assigning a spectrum ofcolors to differing shades of grey. This image may then be convertedback to an analog image (by an ADI board) and displayed for a real timevisualization of differences between an averaged control image andsubsequent images. Moreover, the processed difference image can besuperimposed over the analog image to display specific tissue siteswhere a contract enhancing agent may have a faster uptake, or where anintrinsic signal may be occurring.

Processing speed may be enhanced by adding a real time modular processoror faster CPU chip to the image processor. One example of a real timemodular processor which may be employed in the present invention is a150 RTMP-150 Real Time Modular Processor (Imaging Technology, Woburn,Mass.). The processor may further include an optical disk for storingdigital data, a printer for providing a hard copy of the digital and/oranalog data and a display, such as a video monitor, to permit the userto continuously monitor the comparison data output.

A single chassis may house all of the modules necessary to provideoptical detection and analysis in a format that can be easilyinterpreted, such as an image format, according to the presentinvention. The necessary components, whether or to whatever degreeintegrated, may be installed on a rack that is easily transportable,along with display monitors and peripheral input and output devices.

A preferred high resolution and high performance system comprising aPentaMAX 576×384FT LCD system (by Princeton Instruments Inc., NJ)digitizes the data at the chip and provides a large dynamic range andreduced noise. This system may be interfaced using a PCI-bus to adual-400 Mhz Pentium PC running windows NT. Image analysis algorithmsmay be written in C using Microsoft VisualC++ Version 5.0 compiler. Formore rapid online processing, the data may be routed to dedicatedimaging hardware residing in the PC computer. For example, IM-PCIhardware (by Imaging Technology Inc., Bedford, Mass.) could be used. Onesuch configuration would consist of the following IM-PCI boards andmodules: IM-PCI, AMVS, and a CMALU.

The imaging methods applied to in vivo applications may acquire data atthe surface of an area of interest. As described above, longerwavelengths of emr (in the infrared range) can be used to image areas ofinterest which are deeper in tissue or below overlying tissue. In someareas of the body longer wavelength visible light and near infrared emrcan easily pass through such tissue for imaging. Moreover, if adifference image is created between the image acquired at 500 nm emr andthe image acquired at 700 nm emr, the difference image will show anoptical slice of tissue. Administration of an imaging agent whichabsorbs specific wavelengths of emr can act as a tissue filter of emr toprovide a filter in the area of interest. In this instance, it isdesirable to utilize an imaging agent that remains in the tissue for aprolonged period of time.

In a simple system suitable for assessing cell populations in vivo inanimal or tissue culture models, the systems of the present inventionmay include one or more optical fiber(s) operably connected to an emrsource that illuminates cells or tissue, and another optical fiberoperably connected to an optical detector, such as a photodiode, thatdetects one or more optical properties of the illuminated cells ortissue. The detector may be used to acquire obtain control datarepresenting the “normal” or “background” optical properties of a samplepopulation, and then to acquire subsequent data representing the opticalproperties of the sample population during or following administrationof a test agent or test condition. A physiological challenge and/or astimulus that stimulates a disease or pathological state may beadministered prior to administration of the treatment agent orcondition. The system comprises or is in communication with a datastorage and processing system having information storage and processingcapability sufficient to compare the geometrical and/or opticalproperties of individual cells or cell samples to empirically determinedstandards, or to data acquired at different points in time.

In operation, an area of interest in an in vitro or in vivo cell sampleis illuminated with electromagnetic radiation (emr) and one or a seriesof data points or data sets representing one or more geometrical and/oroptical properties of a spatially resolved area of interest is acquiredduring an interval of “normal” physiological activity. This datarepresents a control, or background data profile for that particularcell sample under those particular physiological conditions. A series ofdata sets is preferably combined, for example by averaging, to obtain acontrol data profile. The control data profile is stored for comparisonwith other data sets. Similarly, control data sets may be collected andstored that represent a background data profile for particular celltypes under specified physiological conditions.

Data sets representing the corresponding geometrical and/or opticalproperty of the sample population at the same, spatially resolved areasof interest, are acquired during a subsequent time period. Formonitoring applications, data may be collected at regular time intervalsto monitor the condition of the cell sample and to detect aberrationsfrom the baseline profile. For screening applications, one or moresubsequent data set(s) is collected during a period followingphysiological activity or inhibition, induced, for example, byintroduction of a test compound or by exposure to a test condition.Physiological activity or inhibition may be induced by a “natural”occurrence such as a seizure or stroke in an animal model, or it may beinduced by administering a paradigm or an agent to an in vitro or invivo cell sample to stimulate changes in geometrical and/or opticalproperties of the cell sample that are indicative of physiologicalactivity or inhibition. During a monitoring interval or stimulation ofan intrinsic physiological response, one or a series of subsequent datasets, representing one or more of the detected geometrical or opticalproperties of the area of interest, is acquired. A series of subsequentdata sets is preferably combined, for example by averaging, to obtain asubsequent data set. The subsequent data set is compared with thecontrol data set to obtain a comparison data set, preferably adifference data set. Comparison data sets are then analyzed for evidenceof changes in geometrical and/or optical properties representative ofphysiological activity or inhibition within the area of interest.

FIG. 1 shows a schematic flow diagram illustrating an exemplary system,as well as exemplary output data of the present invention with referenceto in vitro cell populations. A cell population may comprise cells insuspension at a sparse cell density, or confluent layers of cells, orlayers of cells at other predetermined cell densities, or at issuesample, such as a tissue slice. Maintenance of a wide variety of celland tissue samples under cell culture conditions is well known in theart.

The sample population is placed at a predetermined location on aplatform, such as on a microscope stage. In the example shown in FIG. 1,the sample is an acute rat hippocampal slice maintained in a submergedperfusion chamber. Alternatively, the sample may be cell samplesmaintained in cell culture media in flasks, multiple well plates, andthe like. Multiple well tissue culture plates may be used for highthroughput screening, in combination with an automated stage forpositioning cell samples in individual wells for optical detection atpredetermined intervals. Programmable, automated positioning devices arewell known in the art.

An optical detector (in this case, a CCD camera) is attached to thecamera-port of the microscope. During one or more control period(s) andone or more test period(s), data relating to dimensional and/or opticalproperties of individual cells or of an area of interest in the cellsample are acquired, stored and processed. Acquisition and processing ofdata may be accomplished as described below and in the Examples.

The grey-scale image on the upper right is the unprocessed image of thetissue-slice as viewed by the CCD camera. This slice was thenelectrically stimulated at two different intensities: a low-intensityelectrical stimulus causing a small increase in neuronal and synapticactivation; and a high-intensity electrical stimulus causing a largerincrease in neuronal and synaptic activity.

The image was generated as described below in Example 1. Briefly, animage acquired during the electrical stimulation was subtracted from animage acquired in the control state. This image was then filtered with alow-pass filter, histogram-stretched, and maybe pseudo-colored. If theimage is pseudo-colored, the colors maybe coded to indicate intensity ofactivity-evoked optical change (arrow on color-bar).

The dynamic optical changes represented in these images can also beplotted as a graph (lower right image). Here, each data-point representsthe average change in light-transmission through the small box. A seriesof images, two seconds apart, were acquired and the average value wascalculated for each image and plotted as a point on the graph. Thetissue was electrically stimulated for two seconds at the pointsindicated by the straight lines. The small peak indicates the maximumoptical change induced by the first small electrical stimulation, thelarger peak from the second larger stimulus. The electrical stimulationwas ceased after two seconds and the tissue was allowed to recover. Theplots of the recovery are characteristic of the ion-homeostaticmechanisms of the tissue. Their recovery could be quantified, forexample, by finding the best exponential fits for the recovery periods.

EXAMPLE 1

Sprague-Dawley rats (male and female; 25 to 35 days old) were preparedas described in Aghajanian, A. K. and Rasmussen, K., Synapse 31:331,1989; and Buckmaster, P. S., Strowbridge, B. W., Schwartzdroin, P. A.,J. Neurophysiol. 70:1281, 1993. In most hippocampal slice experiments,simultaneous extracellular field electrode recordings were obtained fromCA1 and CA3 areas. For stimulation-evoked afterdischarge (13 slices, 8animals), the concentration of Mg²⁺ in the bathing medium was reduced to0.9 mM. A bipolar tungsten stimulating electrode was placed on theSchaffer collaterals to evoke synaptically driven field responses inCA1; stimuli consisted to 100 to 300-μs-duration pulses at an intensityof four times population-spike threshold. Afterdischarges were evoked bya 2-s train of such stimuli delivered at 60 Hz. Spontaneousinterictal-like bursts were observed in slices treated with thefollowing modifications or additions to the bathing medium: 10 mM K⁺ (6slices; 4 animals; average, 81 bursts/min), 200 to 300 μM 4-AP (4slices; 2 animals; average, 33 bursts/min), 50 to 100 μg M bicuculine (4slices; 3 animals; average, 14 bursts/min), 0 mM Mg²⁺ [(1 hour ofperfusion) 3 slices; 2 animals; average, 20 bursts/min; (3 hours ofperfusion) 2 slices, 2 animals)], 0 mM Ca²⁺/6 mM KCl and 2 mM EGTA (fourslices, three animals). In all treatments, perfusion withfurosemide-containing medium was begun after a consistent level ofbursting had been established.

For imaging of intrinsic optical signals, the tissue was illuminatedwith a beam of white light (tungsten filament light and lens system;Dedotec USA, Lodi, N.J.) directed through the microscope condenser. Thelight was controlled and regulated (power supply: Lambda Electronics,Melville, N.Y.) to minimize fluctuations and filtered (695 nm long-pass)so that the slice was transilluminated with long wavelengths (red).Image frames were acquired with a charge-coupled device camera(Dage-MTI) at 30 Hz and were digitized at 8 bits with a spatialresolution of 512 by 480 pixels by means of an Imaging Technology Series151 imaging system; gains and offsets of the camera-control box and theanalog-to-digital board were adjusted to optimize the sensitivity of thesystem. Imaging hardware was controlled by a 486-PC-compatible computerrunning software written by D. Hochman and developed with commerciallyavailable software tools (Microsoft's C/C++ Compiler and ImagingTechnology's ITEX library). To increase signal-to-noise ratio, anaveraged image was composed from 16 individual image-frames, integratedover 0.5 s and averaged together. An experimental series typicallyinvolved the continuous acquisition of a series of averaged images overa several minute time period; at least 10 of these averaged images wereacquired as control images before stimulation. Pseudocolored images werecalculated by subtracting the first control image from subsequentlyacquired images and assigned a color lookup table to the pixel values.For these images, usually a linear low-pass filter was used to removehigh-frequency noise and a linear-histogram stretch was used to map thepixel values over the dynamic range of the system. All operations onthese images were linear so that quantitative information was preserved.

FIGS. 2A-2C show the effect of the agent furosemide on stimulationevoked afterdischarge activity in a hippocampal tissue slice comparingthe field response, measurements at an extracellular electrode, andimages highlighting changes in optical properties.

FIG. 2A 1 illustrates that two seconds of electrical stimulation at 60Hz elicited afterdischarge activity. FIG. 2A 2 shows a typicalafterdischarge episode recorded by the extracellular electrode, with thehorizontal arrow indicating the baseline. FIG. 2A 3 shows a map of thepeak change in optical transmission through the tissue evoked bySchaffer collateral stimulation. The region of maximum optical changecorresponds to the apical and basal dendritic regions of CA1 on eitherside of the stimulating electrode. FIG. 2B 1-2BC illustrate responses toelectrical stimulation following 20 minutes of perfusion with mediumcontaining 2.5 mM furosemide. Both the electrical afterdischargeactivity (shown in FIG. 2B 2) and the stimulation-evoked optical changes(shown in FIG. 2BC) were blocked. However, there was a hyperexcitablefield response (multiple population spikes) to the test pulse, asillustrated in FIG. 2B 1. FIGS. 2C1-2C3 illustrate that restoration ofthe initial response pattern was seen following 45 minutes of perfusionwith normal bathing medium.

FIG. 3A illustrates an enlarged grey-scale image of an acute rathippocampal tissue slice, observed using a CCD camera attached to aZeiss upright microscope. FIGS. 3B-3E illustrate enlarged imagesacquired as described above. FIG. 3B illustrates an enlarged imageacquired as described above during the peak optical change induced byelectrical stimulation. The box indicates the field of view shownmagnified in FIGS. 3C, 3D and 3E. FIG. 3C illustrates the peak opticalchange during electrical stimulation when no epileptic activity wasinduced. FIG. 3D illustrates the peak optical change during electricalstimulation that resulted in epileptiform activity. A larger area ofincreased magnitude of changes in optical properties is observed duringepileptiform activity. FIG. 3E illustrates the peak optical changeduring electrical stimulation following treatment with furosemide, whichblocks the epileptiform activity and the intrinsic optical signal.

EXAMPLE 2

This example illustrates optical changes indicative of neuronal activityin a human subject by direct cortical electrical stimulation. Surfaceelectrical recordings (surface EEG, ECOG) were correlated with opticalchanges. Intrinsic optical changes were evoked in an awake patientduring stimulating-electrode “calibration.” Four stimulation trials weresequentially applied to the cortical surface, each stimulation evokingan epileptiform afterdischarge episode. A stimulation trial consistedof: (1) monitoring resting cortical activity by observing the output ofthe recording electrodes for a brief period of time; (2) applying anelectric current via the stimulation-electrodes to the cortical surfaceat a particular current for several seconds; and (3) monitoring theoutput of the recording electrodes for a period of time afterstimulation has ceased.

The cortex was evenly illuminated by a fiber optic emr passing through abeam splitter, controlled by a D.C. regulated power supply (Lambda,Inc.) and passed through a 695 nm longpass filter. Images were acquiredwith a CCD camera (COHU 6500) fitted to the operating microscope with aspecially modified cineadaptor. The cortex was stabilized with a glassfootplate. Images were acquired at 30 Hz and digitized at 8 bits(512×480 pixels, using an Imaging Technology Inc. Series 151 system,Woburn, Mass.). Geometric transformations were applied to images tocompensate for small amounts of patient motion (Wohlberg, DigitalImaging Warping, IEEE Computer Society: Los Alamitos, Calif., 1988).Subtraction of images collected during the stimulated state (e.g.,during cortical surface stimulation, tongue movement or naming) fromthose collected during a control state with subsequent division by acontrol image resulted in percentage difference maps. Raw data (i.e., nodigital enhancement) were used for determining the average opticalchange in specified regions (average sized boxes was 30×30 pixels or150-250 μm ²). For pseudocolor images, a linear low pass filter removedhigh frequency noise and linear histogram transformations were applied.Noise was defined as the standard deviation of fluctuations insequentially acquired control images as 0.003-0.009.

A series of images (each image consisting of an average of 128 framesacquired at 30 Hz) were acquired during each of the four stimulationtrials. A current of 6 mA was used for the first three stimulationtrials, and 8 mA for the fourth. After a sequence of 3-6 averagedcontrol images were acquired, a bipolar cortical stimulation current wasapplied (either 6 mA or 8 mA) until epileptiform after dischargeactivity was evoked (as recorded by the surface electrode). Images werecontinuously acquired throughout each of the four stimulation trials.

The percentage change in absorption of light for each pixel wascalculated for each image acquired during the four stimulation trials.The average percentage changes over the four areas (indicated by thefour square regions marked in FIG. 4A) were plotted graphically in FIGS.4B, 4C, and 4D for comparison and analysis of the dynamic changesoccurring in these four spatial areas.

The optical changes between the stimulating electrodes (Site #1, FIG.4A) and near the recording electrode (Site #3) showed a graded responseto the intensity and duration of each afterdischarge episode (FIG. 4B).The spatial extent of the epileptiform activity was demonstrated bycomparing a baseline image collected before stimulation to thoseobtained immediately after stimulation. The intensity and spread of theoptical changes were much less following Stimulation #2 (shortest, leastintense afterdischarge episode) than after Stimulation #4 (longest, mostintense afterdischarge episode).

When the optical changes were below baseline, the surface EEG recordingsdid not identify epileptiform activity (n=3 patients). At Site #3, theoptical changes after stimulation were below baseline. However, duringthe fourth stimulation, the epileptiform activity spread into the areaof Site #3 and the optical signal did not go below baseline until later.This negative optical signal likely represents inhibited neuronalpopulations (an epileptic inhibitory surround), decreased oxygendelivery, or blood volume shunted to activated regions.

FIG. 4B shows plots of the percent optical change per second in thespatial regions of Boxes 1 and 3 (as labeled in FIG. 4A). For bothregions, the peak change is during the fourth stimulation trial (at 8mA), in which the greatest amount of stimulating current had induced themost prolonged epileptiform afterdischarge activity. The changes withinBox 3 were greater and more prolonged than those of Box 1. Box 3 wasoverlying the area of the epileptic focus.

FIG. 4C shows plots of the percent optical change per second in thespatial regions of Boxes 1 and 4 (as labeled in FIG. 4A). Box 1 overlaysan area of cortical tissue between the two stimulating electrodes, andBox 4 overlays a blood vessel. The optical changes within box 4 are muchlarger and in the opposite direction of Box 1. Also, these changes aregraded with the magnitude of stimulating current and afterdischargeactivity. The changes in Box 4 are most likely due to changes of theblood-flow rate within a blood vessel.

FIG. 4D shows plots of the percent optical change absorption per secondin the spatial regions of boxes 1 and 2 (as labeled in FIG. 4A). Notethat although these two areas are nearby each other, their opticalchanges are in the opposite direction during the first three stimulationtrials using 6 mA current. The negative going changes within the regionof Box 2 indicate that the methods and apparatus of the presentinvention may be used to monitor inhibition of physiological changes, aswell as excitation.

FIG. 5 shows percentage difference images representative of varioustimes during two of the stimulation trials described above. The topthree images (5A2, 5B2, and 5C2) are from Stimulation Trial 2, where 6mA cortical stimulation evoked a brief period of afterdischarge. Theseare compared to the bottom three images (5A4, 5B4, and 5C4), which arefrom Stimulation Trial 4, showing the optical changes evoked by corticalstimulation at 8 mA. FIGS. 5A2 and 5A4 compare control images duringrest. FIGS. 5B2 and 5B4 compare the peak optical changes occurringduring the epileptiform afterdischarge activity. FIGS. 5C2 and 5C4compare the degree of recovery 20 seconds after the peak optical changeswere observed. The magnitude of optical change is indicated by thegrey-scale changes. Each image maps an area of cortex approximately 4 cmby 4 cm.

FIGS. 6A-6H show eight percentage difference images from StimulationTrial 2, as described above. Each image is integrated over a two secondinterval. The focal area of greatest optical change is in the center ofimages 3C, 3D, and 3E, indicating the region of greatest corticalactivity. This region is the epileptic focus. The magnitude of opticalchange is indicated by the grey-scale bar on the right side of theFigure. The arrow beside this grey-scale indicates the direction ofincreasing amplitude. Each image maps an area of cortex approximately 4cm by 4 cm.

FIGS. 7A-7H illustrate a real-time sequence of dynamic changes ofstimulation-evoked optical changes in human cortex. FIG. 4, panels 4Athrough 4H, show eight consecutive percentage difference images. Eachimage is an average of 8 frames (<¼ second per image). The magnitude ofoptical change is indicated by the grey-scale changes. Each image mapsto an area of cortex that is approximately 4 cm by 4 cm. This figuredemonstrates that the methods and apparatus of the present invention canbe used to acquire, in real time, data reflecting dynamic changes ofoptical properties that reflect physiological changes.

EXAMPLE 3

Stimulation mapping of the cortical surface was performed on awake humanpatients under local anesthesia to identify sensory/motor cortex andBroca's areas. The illumination source and optical detection device andprocessing techniques used were the same as those described in Example2. During three “tongue wiggling” trials, images were averaged (32frames, 1 sec) and stored every 2 seconds. A tongue wiggling trialconsisted of acquiring 5-6 images during rest, then acquiring imagesduring the 40 seconds that the patient was required to wiggle his tongueagainst the roof of his mouth, and then to continue acquiring imagesduring a recovery period. The same patient was then required to engagein a “language naming” trial. A language naming trial consisted ofacquiring 5-8 images during rest (control images—the patient silentlyviewing a series of blank slides), then acquiring images during theperiod of time that the patient engaged in the naming paradigm (naming aseries of objects presented with a slide projector every 2 seconds,selected to evoke a large response in Broca's area), and finally aseries of images during the recovery period following the time when thepatient ceased his naming task (again viewing blank slides whileremaining silent). The results are shown in FIGS. 8 and 9.

FIGS. 8A1-8BC illustrate functional mapping of human language (Broca'sarea) and tongue and palate sensory areas in an awake human patient asdescribed in Example 3. Images 8A1 and 8B1 are grey-scale images of anarea of human cortex, with left being anterior, right-posterior,top-superior, and the Sylvan fissure on the bottom. The two asterisks on8A1, 8B11, 8A2, and 8B2 serve as reference points for these images. Thescale bars in the lower right corner of 8A1 and 8B1 are equal to 1 cm.In 8A1, the numbered boxes represent sites where cortical stimulationwith electrical stimulating electrodes evoked palate tingling (1),tongue tingling (2), speech arrest-Broca's areas (3,4) and no response(11, 12, 17, 5, 6-7 premotor). Image 8A2 is a percentage differencecontrol image of the cortex during rest in one of the tongue wigglingtrials. The grey-scale bar on the right of 8A2 shows the relativemagnitude of the grey values associated with images 8A2, 8A3, 8B2 and8B3. Image 8A3 is a percentage difference map of the peak opticalchanges occurring during one of the tongue wiggling trials. Areasidentified as tongue and palate sensory areas by cortical stimulationshowed a large positive change. Suppression of baseline noise insurrounding areas indicated that, during the tongue wiggling trials,language-motor areas showed a negative-going optical signal. Image 8B2is percentage difference control image of the cortex during one of thelanguage naming trials. Image 8B3 is a percentage difference image ofthe peak optical change in the cortex during the language naming task.Large positive-going signals are present in Broca's area. Negative-goingsignals are present in tongue and palate sensory areas.

FIG. 9 shows time course and magnitude plots of dynamic optical changesin human cortex evoked in tongue and palate sensory areas and in Broca'sarea (language). This figure shows the plots of the percentage change inthe optical absorption of the tissue within the boxed regions shown inFIG. 8, images 8A1 and 8B1, during each of the three tongue wigglingtrials and one of the language naming trials (see description of FIG.8). FIG. 9A shows the plots during the three tongue wiggling trialsaveraged spatially within the Boxes 1, 2, 3, and 4 as identified in FIG.8A 1. FIG. 9B shows the plots during one of the language naming trialsaveraged spatially within the Boxes 1-7 and 17.

These results agree with those data reported by Lee, et al. (Ann.Neurol. 20:32, 1986), who reported large electrical potentials in thesensory cortex during finger movement. The magnitude of the opticalchanges in the sensory cortex during tongue movement (10-30%) parallelssensory/motor cortex studies where cerebral blood flow increases 10-30%during motor tasks (Colebatch et al., J. Neurophysiol. 65:1392, 1991).Further, utilizing Magnetic Resonance Imaging (MRI) of blood volumechanges in human visual cortex during visual stimulation, investigatorshave demonstrated increases of up to 30% in cerebral blood volume(Belliveau et al., Science 254:716, 1991).

Optical images were obtained from this same cortical region (i.e., areaof interest) while the patient viewed blank slides and while namingobjects on slides presented every two seconds. Percentage differencemaps obtained during naming showed activation of the premotor area. Thesites of speech arrest and palate tingling were identified by surfacestimulation and demonstrate optical signals going in the oppositedirection. The area of activation was clearly different from that evokedby tongue movement without speech production. The optical images ofpremotor cortex activation during naming were in similar locations tothe cortical areas identified in PET single word processing studies(Peterson, et al., Nature 331:585, 1991; and Frith et al., J.Neuropsychologia 29:1137, 1991). The optical changes were greatest inthe area of the cortex traditionally defined as Broca's area and not inareas where electrical stimulation caused speech arrest.

EXAMPLE 4

Areas of interest can be imaged through intact tissues, such as bone,dura, muscle, connective tissue and the like. FIGS. 10A-10D illustrateidentification of a brain tumor through the intact cranium using opticalimaging techniques of the present invention.

FIG. 10A is a grey-scale image of the cranial surface of a rat. Thesagittal suture runs down the center of the image. Box 1 lays over thesuspected region of brain tumor, and Box 2 lays over normal tissue. FIG.10B is a difference image one second after indocyanine green dye hadbeen intravenously injected into the animal. The region containing tumortissue became immediately visible through the intact cranium. FIG. 10Cshows that five seconds after dye injection the dye can be seen toprofuse through both normal and tumor tissue. FIG. 10D shows that oneminute after dye injection, the normal tissue had cleared the dye, butdye was still retained in the tumor region. The concentration of dye inthe center of this difference image was dye circulating in the sagittalsinus. Dynamic changes in optical properties of cell populations andtissue may likewise be imaged in vivo through other intact tissues, suchas bone, dura, muscle, connective tissue, and the like.

1. A method for assessing the physiological condition of a biologicalmaterial, comprising: maintaining at least one sample population of thebiological material in one of the following systems: a cell culturesystem; a tissue culture system; an organ culture system; and an intactorganism: acquiring test data relating to one or more geometrical oroptical properties of the sample population; and comparing the test dataacquired to comparison data relating to one or more geometrical oroptical properties of a comparison cell population, the comparison datarepresenting one or more geometrical or optical properties of thecomparison cell population in a predetermined physiological state,whereby changes in the one or more geometrical or optical propertiesreflected in the test data compared to the comparison data representchanges in the physiological state of the sample population.
 2. A methodof claim 1, additionally comprising acquiring multiple test data setsrelating to one or more geometrical or optical properties at multiple,predetermined spatial locations in the sample population.
 3. A method ofclaim 1, additionally comprising exposing the sample population to aphysiological challenge prior to acquiring the test data.
 4. A method ofclaim 3, additionally comprising acquiring control data relating to theone or more geometrical properties of the sample population prior toexposing the sample population to a physiological challenge.
 5. A methodof claim 4, additionally comprising comparing the test data to thecontrol data to assess changes in the one or more geometrical or opticalproperties of the sample population representing changes in thephysiological state of the sample population.
 6. A method of claim 1,wherein the comparison data is derived from empirically determinedcontrols.
 7. A method of claim 3, wherein the physiological challenge isselected from the group consisting of: exposure to a test agent, a testcondition, a drug, a hormone, a biological agent, a toxin, an infectiousagent, radiation, chemotherapy, deprivation of a metabolite or nutrient,electrical stimulation, inflammatory agent, oncogen.
 8. A method ofclaim 1, additionally comprising maintaining multiple sample populationsin an in vitro culture system.
 9. A method of claim 1, wherein the testdata acquired relates to one or more optical properties selected fromthe group consisting of: reflection, refraction, diffraction,absorption, scattering, birefringence, refractive index and Kerr effect.10. A method according to claim 1, wherein the biological material is aviable sample population maintained in a cell culture system.
 11. Amethod according to claim 1, wherein the biological material is aviable, intact organism.
 12. A system for assessing the physiologicalcondition of a biological material, comprising: a platform forsupporting an optically transparent container of biological material; anillumination source for illuminating the biological material; an opticaldetector for acquiring test data relating to a geometrical or opticalproperty of the biological material; and a data storage and analysisdevice for storing data relating to a geometrical or optical property ofthe biological material, and comparing test data with a control dataprofile to generate a comparison data set relating to geometrical oroptical property of the biological material;
 13. A system of claim 12,wherein the platform is an automated stage operated by a control devicecapable of locating and moving the stage to predetermined x-ycoordinates.
 14. A system of claim 12, additionally comprising a dataoutput device capable of displaying the comparison data in a visualformat.
 15. A system of claim 14, wherein the data output device iscapable of displaying comparison data in a graphical or image format.16. A system of claim 12, wherein the optical detector is a chargecoupled device (CCD).